Solid state nanopore bio-sensing is emerging as a rapid single molecule sensing technique (Branton et al., 2008, Nature Biotechnology, 26, 1146; Dekker, 2007, Nature Nanotechnology 2, 209). Conceptually, a single nanometer size aperture located on a membrane can detect electrophoretically driven biomolecules translocation in a high throughput manner, revealing localized information of the analyte. However, the formation of single nanopores relies heavily on expensive instrumentation, i.e., Transmission Electron Microscope (TEM) and well trained TEM user, which renders it still confined to laboratory use since this nanopore fabrication process is time-consuming, expensive, not scalable and hard to control at the nm scale.
Further, high costs of the TEM use, coupled with its high initial investment and the time consuming pore drilling process (1 hour machine and operator time per device) limit the more extensive application of solid state nanopores in the bio-sensing field. In addition, not all TEM drilled nanopores are hydrophilic and functional for the sensing of biomolecules. In addition, interaction with the high energy electron beam can cause damage especially when dealing with membranes in 2 D materials.
Many efforts, such as chemical wet-etching of silicon (Park et al., 2007, Small 3, 116-119) or polyethylene terephthalate film (Siwy et al., 2002, Physical review letters 89, 198103) have been carried out towards mass production of nanopores. Recently, a facile method has been reported using dielectric breakdown to make individual nanopores (3-30 nm diameter) on insulating silicon nitride membranes (5-30 nm thick) without the need of TEM_ENREF_8 (Kwok et al., 2014, Plos One 9, doi:10.1371/journal.pone.0092880, WO 2013/167952) for the in-situ forming of nanopores. However, those techniques based on dielectric breakdown need to apply high voltages pulses to the membranes which should be as short as possible for trying to monitor the nanopore diameter during its formation (WO 2014/144818). When reaching dielectric breakdown, the process of pore forming becomes rather uncontrollable which is problematic for reproducibility and quality control of the nanopore size, especially when formed in-situ in a nanopore bio-sensing device, thereby leading to important production waste if the quality of the pore does not correspond in fine to the prescribed parameters.
Atomically thin nanopore membranes, graphene (Garaj et al., 2010, Nature, 467, 190) and molybdenum disulphide (MoS2) (Feng et al., 2015, Nature Nanotechnology, 10, 1070) have drawn much attention recently due to their unprecedented single nucleotide resolution and holds promise as a candidate for so called 3rd generation DNA sequencers. Therefore, if fabrication of nanostructures with sub-nanometer, or even single-atom precision has been a long-term goal for nanotechnology in general, there is now a raise of interest for those thin nanopore membranes and an increased need for cost-effective and reliable techniques for nanopore formation in those membranes.
In particular, since the differentiation of biomolecules relies strongly on the pore diameter, there is a high need for developing methods allowing controllable nanopore fabrication, which would enable mass production nanopore in 2D membranes such as MoS2 even below 4 nm with atomic precision.